Leishmania

Dr N P SinghProfessor

Deptt. Of Microbiology

Leishmania

Phylum

Order

Family

Genus

Sarcomastigophora

Kinetoplastida

Trypanosomatidae

Leishmania

• Transmitted to the mammalian hosts by the bite of infected sandflies, Phlebotomus and Lutzomyia

• Currently, leishmaniasis occurs in 4 continents and is considered to be endemic in 88 countries, 72 of which are developing countries: 90% of all VL: Bangladesh, Brazil, India, Nepal and

Sudan 90% of all MCL: Bolivia, Brazil and Peru 90% of all CL : Afghanistan, Brazil, Iran, Peru, Saudi

Arabia and Syria

• Annual incidence: 1- 1.5 million cases of CL

: 500,000 cases of VL

• Prevalence: 12 million people

• Population at risk: 350 million

(WHO, 2010)

SITUATION IN INDIA• 40-50% of global burden

(Bora 1999, Natl Med J India)

• Surveillance being done by NVBDCP

• INDIA: 15538 cases and 47 deaths by VL (2010)

• Endemic states in Eastern India: Bihar, Jharkhand, West Bengal, Uttar Pradesh

• Estimated 165.4 million population at risk in 4 states

(NVBDCP, 2010)

• On the basis of development, divided in two genera:

Subgenus Leishmania: in the anterior part of alimentary tract of sandfly

Subgenus Viannia: midgut and hindgut of sandfly

Leishmania L. major complex (L. major) Old World

L. tropica complex (L. tropica, L. killicki)

L. aethiopica complex (L. aethiopica)

L. donovani complex (L. donovani, L. infantum)

L. donovani complex (L. chagasi) New World

L. mexicana complex (L. mexicana, L. venezuelensis, L. garnhami, L. amazonensis, L.pifanoi)

Viannia L. braziliensis complex (L. braziliensis, L. peruviana, L. columbiensis, L. lainsoni)

L. guyanensis complex (L. guyanensis, L. panamensis)

Leishmania Parasites and DiseasesDisease SPECIES

Cutaneous leishmaniasis)CL(

Leishmania tropicaLeishmania major

Leishmania aethiopicaLeishmania mexicana

Mucocutaneous leishmaniasis (MCL)

Leishmania braziliensis

Visceral leishmaniasis)VL(

Leishmania donovaniLeishmania infantumLeishmania chagasi

Promastigote– Insect– Motile– Midgut

Amastigote– Mammalian

stage– Non-motile– Intracellular

Digenetic Life Cycle

INFECTION

Sub-clinical or inapparent infection

Recovery DeathImmune to reinfection Concurrent

infection PKDL

LEISHMANIASIS

• Vector borne disease

• A wide clinical spectrum VISCERAL PKDL DIFFUSE CUTANEOUS CUTANEOUS MUCOCUTANEOUS

VL - Clinical Manifestation

• Variable - Incubation 3-100+ weeks• Lowgrade fever• Hepato-splenomegaly• Bone marrow hyperplasia• Anemia, Leucopenia & Cachexia• Hypergammaglobulinnemia• Epistaxis , Proteinuria, Hematuria

• Most severe form of the disease, may be fatal if left untreated

• Usually associated with fever, weight loss, and an enlarged spleen and liver

• Anemia (low RBC), leukopenia (low WBC), and thrombocytopenia (low platelets) are common

• Lymphadenopathy may be present

• Visceral disease from the Middle East is usually milder with less specific findings than visceral leishmaniasis from other areas of the world

Post Kala Azar Dermal Leishmaniasis

• Normally develops <2 years after recovery

• Recrudescence

• Restricted to skin

• Rare but varies geographically

Cutaneous Leishmaniasis• Most common form• Characterized by one or more sores, papules or

nodules on the skin• Sores can change in size and appearance over time• Often described as looking somewhat like a

volcano with a raised edge and central crater• Sores are usually painless but can become painful if

secondarily infected• Swollen lymph nodes may be present near the

sores (under the arm if the sores are on the arm or hand…)

Cutaneous Leishmaniasis• Most sores develop within a few weeks of the sandfly

bite, however they can appear up to months later

• Skin sores of cutaneous leishmaniasis can heal on their own, but this can take months or even years

• Sores can leave significant scars and be disfiguring if they occur on the face

• If infection is from L. tropica it can spread to contiguous mucous membranes (upper lip to nose)

Mucocutaneous Leishmaniasis

• Occurs with Leishmania species from Central and South America

• Very rarely associated with L. tropica which is found in the Middle East- This type occurs if a cutaneous lesion on the face spreads

to involve the nose or mouth - This rare mucosal involvement may occur if a skin lesion near the mouth or nose is not treated• May occur months to years after original skin lesion• Hard to confirm diagnosis as few parasites are in the lesion• Lesions can be very disfiguring

DIAGNOSIS

Direct evidence: Demonstration of Leishmania

Specimens that may be collected • Splenic aspirate and biopsy• Liver biopsy• Bone marrow (Sternum or iliac crest)• FNAC and biopsy• Blood buffy coat• Tegumantary leishmaniasis- dermal scrapings, sections

from skin biopsy

DEMONSTRATION OF Leishmania AMASTIGOTES/ L.D. BODIES

MICROSCOPY

CULTURECulture media for axenic culture• SOLID MEDIUM

NNN medium Evan’s modified Tobie’s medium

• LIQUID MEDIA Schneider’s Drosophila medium Grace’s insect tissue culture medium

DEMONSTRATION OF Leishmania PROMASTIGOTES

Animal inoculation• Golden hamsters inoculated intraperitoneally

Promastigotes as seen in artificial culture medium

IMMUNOLOGICAL METHODS

• Aldehyde (formol gel) Test (Napier)

• Antimony test (Chopra)

• Complement fixation test with W.K.K Ag

Indirect Fluorescent Antibody test

• Detection of anti-leishmanial antibody using fixed promastigotes

• Demonstrated in the very early stages of infection and undetectable six to nine months after cure

• Titers >1:20 are significant and above 1:128 are diagnostic

• Cross reaction with trypanosomal sera (overcome by using Leishmania amastigotes as the antigen instead of the promastigotes)

Direct Agglutination Test

• Use of whole, stained promastigotes either as a suspension or in a freeze-dried form.

• The freeze-dried form is heat stable

• Utilized for field purposes

• Relative long incubation time of 18 hours

• Need for serial dilutions of serum

• No prognostic value• Remain positive for several

years after cure

Modifications of DAT

• Fast Agglutination Screening Test

(Schoone et al, 2001) 

Need of only 1 serum dilution Rapid: results available in less than 3 hours

• EasyDAT method

(Gomez-Ochoa et al, 2003, Clin Diagn Lab Immunol)

ELISA BASED ASSAYS

Many antigens have been explored for the diagnosis of leishmaniasis:

• Whole soluble antigens (Ld-ESM—Excretory, secretory and metabolic antigen by L.donovani)

• Purified antigens such as fucose- mannose• Defined, synthetic peptides• Recombinant antigens

rGBP (L.major protein encoding a hydrophilic protein) rORFF (L. infantum) gp63 rK39 rK26, rK9 rKE16

rK39• Rapid dipstick test

• Based on the recombinant k39 protein, a 39-amino acid cloned in Escherichia coli, from the C terminus of the kinesin protein of Leishmania major in India

• Case definition for enrolling a subject

“A case presenting to a clinician with a fever of more than two weeks duration, with splenomegaly and not responding to the full course of anti-malarials”

• Sensitivity-100%, Specificity-97% (NVBDCP, 2010)

• Not to be used in the following cases: Kala-azar relapses In cases of kala-azar re-infection Kala-azar and HIV co-infection

• This test has been incorporated in NVBDCP for the diagnosis of VL in India.

Prevention• Suppress the reservoir: dogs, rats, gerbils, other small

mammals and rodents

• Suppress the vector: Sandfly• Critical to preventing disease in stationary troop populations

• Prevent sandfly bites: Personal Protective Measures• Most important at night• Sleeves down• Insect repellent w/ DEET• Permethrin treated uniforms• Permethrin treated bed nets

TreatmentCutaneous and Mucocutaneous

• Antimony (Pentostam®, Sodium stibogluconate) is the drug of choice• Given under an experimental protocol at Walter Reed Army Medical

Center (WRAMC)• 20 days of intravenous therapy • Available at WRAMC for all branches of the military• Requires patient to come to WRAMC

• Fluconazole may decrease healing time in L. major infection• Biopsy and culture to determine species is required• Six weeks of therapy is needed

Visceral Leishmaniasis

• Liposomal amphotericin-B (AmBisome®) is the drug of choice• 3 mg/kg per day on days 1-5, day 14 and day 21

• Pentostam® is an alternative therapy• 28 days of therapy is required

• Although AmBisome® is widely available, the difficulty of accurate diagnosis and the potential severity of visceral infection suggest possible patients be referred to the Leishmania Treatment Center at WRAMC for maximal diagnostic efficiency

Vaccine

• There is as yet no effective vaccine for prevention of any form of leishmaniasis.

• first generation vaccine was prepared using whole killed parasites combined or not with BCG.

• Live: including new genetically modified constructs• 1st generation vaccines: whole killed parasite

with/without adjuvants or fractions of the parasite• 2nd generation vaccines: recombinant proteins, DNA

vaccines & combinations